title: Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector-mediated zinc finger nuclease expression in human-induced pluripotent stem cells.
abstract: BACKGROUND
  The AAVS1 locus is viewed as a 'safe harbor' for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs).
  
  
  METHODS
  We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene.
  
  
  RESULTS
  Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus.
  
  
  CONCLUSIONS
  Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Felix Chang
authors.initials: FC
authors.lastname: Tay
authors.forename: Wee Kiat
authors.initials: WK
authors.lastname: Tan
authors.forename: Sal-Lee
authors.initials: SL
authors.lastname: Goh
authors.forename: Chrishan J A
authors.initials: CJ
authors.lastname: Ramachandra
authors.forename: Cia-Hin
authors.initials: CH
authors.lastname: Lau
authors.forename: Haibao
authors.initials: H
authors.lastname: Zhu
authors.forename: Can
authors.initials: C
authors.lastname: Chen
authors.forename: Shouhui
authors.initials: S
authors.lastname: Du
authors.forename: Rui Zhe
authors.initials: RZ
authors.lastname: Phang
authors.forename: Mohammad
authors.initials: M
authors.lastname: Shahbazi
authors.forename: Weimin
authors.initials: W
authors.lastname: Fan
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
mesh_terms: D016367:Baculoviridae / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D019076:Transgenes; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004720:Endonucleases
keywords: baculoviral transduction; cassette exchange; homologous recombination; iPS cells; zinc finger nuclease